Method of Increasing the Function of an AAV Vector

ABSTRACT

A method of correcting singletons in a selected AAV sequence in order to increasing the packaging yield, transduction efficiency, and/or gene transfer efficiency of the selected AAV is provided. This method involves altering one or more singletons in the parental AAV capsid to conform the singleton to the amino acid in the corresponding position(s) of the aligned functional AAV capsid sequences.

BACKGROUND OF THE INVENTION

Adeno-associated virus (AAV), a member of the Parvovirus family, is asmall nonenveloped, icoshedral virus with single-stranded linear DNAgenomes of 4.7 kilobases (kb) to 6 kb. AAV is assigned to the genus,Dependovirus, because the virus was discovered as a contaminant inpurified adenovirus stocks. AAV's life cycle includes a latent phase atwhich AAV genomes, after infection, are site specifically integratedinto host chromosomes and an infectious phase in which, following eitheradenovirus or herpes simplex virus infection, the integrated genomes aresubsequently rescued, replicated, and packaged into infectious viruses.The properties of non-pathogenicity, broad host range of infectivity,including non-dividing cells, and potential site-specific chromosomalintegration make AAV an attractive tool for gene transfer.

AAV vectors have been described for use as delivery vehicles for boththerapeutic and immunogenic molecules. To date, there have been severaldifferent well-characterized AAVs isolated from human or non-humanprimates (NHP).

Recently, investigators have described a large number of AAVs ofdifferent sequences [G. Gao, et al., Proc Natl Acad Sci USA,100(10):6081-6086 (May 13, 2003); US-2003-0138772-A1 (Jul. 24, 2003)]and characterized these AAVs into different serotypes and clades [G.Gao, et al., J. Virol., 78(12):6381-6388 (June 2004); InternationalPatent Publication No. WO 2005/033321]. It has been reported thatdifferent AAVs exhibit different transfection efficiencies, and alsoexhibit tropism for different cells or tissues.

What is desirable are AAV-based constructs for delivery of heterologousmolecules to different cell types.

SUMMARY OF THE INVENTION

The present invention provides a method of improving vectors derivedfrom AAV which are non-functional and/or which perform weakly.

In one aspect, the method provides a method for correcting singletons ina selected AAV sequence in order to increase the packaging yield,transduction efficiency, and/or gene transfer efficiency of the selectedAAV. This method involves altering one or more singletons in theparental AAV capsid to conform the singleton to the amino acid in thecorresponding position(s) of the aligned functional AAV capsidsequences.

In another aspect, the invention provides modified AAV sequences, i.e.,sequences with one or more singletons eliminated.

In yet another aspect, the invention provides AAV vectors havingmodified AAV capsids according to the present invention.

Other aspects and advantages of the invention will be readily apparentfrom the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating in vitro 293 transduction of singletoncorrected AAV vectors. Singleton corrections are indicated after thevector name with—if present—a number to indicate the number of mutationsperformed.

FIGS. 2A-2C are line graphs illustrating the titration of AAV vectors on293 cells at multiplicities of infection ranging from 10¹ to 10⁴, with acomparison between parent rh.8 and singleton-corrected rh.8 (rh.8R) inFIG. 2A, parent rh.37 and modified rh.37 (FIG. 2B), and AAV2 and AAV8 inFIG. 2C. As a control, a similar titration of AAV2 and AAV2/8 eGFPexpressing vector is presented. Percent (%) of eGFP positive cells ispresented on the Y-axis and was assayed by flow cytometry.

FIG. 3 is a phylogenetic tree of AAV sequences, which indicates theirphylogenetic relationship and clades.

FIGS. 4A-4K are an alignment of the nucleic acid sequences of the capsidprotein (vp1) of AAV2 [SEQ ID NO:7], cy.5 [SEQ ID NO:8], rh.10 [SEQ IDNO: 9], rh.13 [SEQ ID NO: 10], AAV1 [SEQ ID NO: 11], AAV3 [SEQ ID NO:12], AAV6 [SEQ ID NO: 13], AAV7 [SEQ ID NO: 14], AAV8 [SEQ ID NO: 15],hu.13 [SEQ ID NO:16], hu.26 [SEQ ID NO: 17], hu.37 [SEQ ID NO: 18],hu.53 [SEQ ID NO: 19], rh.39 [SEQ ID NO: 20], rh.43 [SEQ ID NO: 21] andrh.46 [SEQ ID NO: 22].

FIGS. 5A-5D are an alignment of the amino acid sequences of the capsidprotein (vp1) of AAV2 [SEQ ID NO: 23], cy.5 [SEQ ID NO: 24], rh.10 [SEQID NO: 25], rh.13 [SEQ ID NO: 26], AAV1 [SEQ ID NO: 27], AAV3 [SEQ IDNO: 28], AAV6 [SEQ ID NO: 29], AAV7 [SEQ ID NO: 30], AAV8 [SEQ ID NO:31], hu.13 [SEQ ID NO: 32], hu.26 [SEQ ID NO: 33], hu.37 [SEQ ID NO:34], hu.53 [SEQ ID NO: 35], rh.39 [SEQ ID NO: 36], rh.43 [SEQ ID NO: 37]and rh.46 [SEQ ID NO: 38].

FIG. 6A-6B are an alignment of the amino acid sequences of the capsidprotein (vp1) of rh.13 [SEQ ID NO: 26], rh2 [SEQ ID NO: 39], rh.8 [SEQID NO: 41], hu.29 [SEQ ID NO: 42], and rh.64 [SEQ ID NO: 43].

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for improving the function of anAAV vector. The present invention is particularly well suited to improvethe packaging yield, transduction efficiency, and/or gene transferefficiency of an AAV vector having a capsid of an AAV which contains oneor more singletons. The invention further provides novel AAV capsidsequences identified and prepared according to the method of theinvention.

As used throughout this specification and the claims, the terms“comprising” and “including” are inclusive of other components,elements, integers, steps and the like. Conversely, the term“consisting” and its variants are exclusive of other components,elements, integers, steps and the like.

Singleton Method of Invention

As used herein, the term “singleton” refers to a variable amino acid ina given position in a selected (i.e., parental) AAV capsid sequence. Thepresence of a variable amino acid is determined by aligning the sequenceof the parental AAV capsid with a library of functional AAV capsidsequences. The sequences are then analyzed to determine the presence ofany variable amino acid sequences in the parental AAV capsid where thesequences of the AAV in the library of functional AAVs have completeconservation. The parental AAV sequence is then altered to change thesingleton to the conserved amino acid identified in that position in thefunctional AAV capsid sequences. According to the present invention, aparental AAV sequence may have 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 2singletons. A parental AAV sequence may have more than 6 singletons.

Once modified, the modified AAV capsid can be used to construct an AAVvector having the modified capsid. This vector can be constructed usingtechniques known to those of skill in the art.

The AAV selected for modification according to the invention method isone for which it is desirable to increase any one or more of thefollowing three functional properties of AAV, i.e., packaging into theviral particle having the capsid of the selected AAV sequence,increasing transduction efficiency, or increasing gene transferefficiency as compared to the parental AAV. For example, the parentalAAV may be characterized by having a lower packaging efficiency thanother, closely related AAV. In another example, the parental AAV mayhave a lower transduction efficiency as compared to closely relatedAAVs. In another example, the parental AAV may have a lower genetransfer efficiency (i.e., a lower ability to deliver a target moleculein vivo) as compared to closely related AAVs. In other examples, theparental AAV is characterized by adequate function in each of thesecategories, but increased function one or more of these areas isdesired.

Thus, the invention provides a library of functional AAVs, the sequencesof which are to be compared to the selected (parental) AAV. Suitably,the library contains AAVs which have a desired function which istargeted for improvement in the selected parental AAV. In other words,each of the sequences in the library of functional AAVs is characterizedby a desired level of packaging ability, a desired level of in vitrotransduction efficiency, or a desired level of in vivo gene transferefficiency (i.e., the ability to deliver to a target selected targettissue or cell in a subject). The functional AAVs which compose thelibrary may individually have one, two or all of these functionalcharacteristics. Other desired functions for the library may be readilydetermined by one of skill in the art.

In one embodiment, a functional AAV is an AAV characterized by theability to produce viral particles with equivalent or greater packagingand transduction efficiency as any one of AAV1, AAV2, AAV7, AAV8 orAAV9. Function may be assessed in a pseudotyping setting with AAV2 repand AAV2 ITRs. Thus, an altered parental AAV can be constructed usingconventional techniques and the AAV vector is considered functional ifvirus is produced from the parental AAV at titers of at least 50% whencompared to production of AAV2. Further, the ability of AAV to transducecells can be readily determined by one of skill in the art. For example,a parental AAV can be constructed such that it contains a marker genewhich allows ready detection of virus. For example, the AAV containseGFP or another gene which allows fluorescent detection. Where the AAVcontains CMV-eGFP, when the virus produced from the altered parental AAVcapsid is transduced into 293 cells at a multiplicity of infection of10⁴, function is demonstrated where transduction efficiency is greaterthan 5% GFP fluorescence of total cells in a context where the cellswere pretreated with wild-type human adenovirus type 5 at a multiplicityof infection of 20 for 2 hours.

Suitably, a library is composed of at least three or at least fourfunctional AAV capsid sequences which represent at least two differentclades. Preferably, at least two sequences from each of the representedclades is included in the library. In certain embodiments, three, four,five, six, or more clades are represented.

A “clade” is a group of AAV which are phylogenetically related to oneanother as determined using a Neighbor-Joining algorithm by a bootstrapvalue of at least 75% (of at least 1000 replicates) and a Poissoncorrection distance measurement of no more than 0.05, based on alignmentof the AAV vp1 amino acid sequence.

The Neighbor-Joining algorithm has been described extensively in theliterature. See, e.g., M. Nei and S. Kumar, Molecular Evolution andPhylogenetics (Oxford University Press, New York (2000). Computerprograms are available that can be used to implement this algorithm. Forexample, the MEGA v2.1 program implements the modified Nei-Gojoborimethod. Using these techniques and computer programs, and the sequenceof an AAV vp1 capsid protein, one of skill in the art can readilydetermine whether a selected AAV is contained in one of the cladesidentified herein, in another lade, or is outside these clades.

While the AAV clades are based primarily upon naturally occurring AAVvp1 capsids, the clades are not limited to naturally occurring AAV. Theclades can encompass non-naturally occurring AAV, including, withoutlimitation, recombinant, modified or altered, chimeric, hybrid,synthetic, artificial, etc., AAV which are phylogenetically related asdetermined using a Neighbor-Joining-algorithm at least 75% (of at least1000 replicates) and a Poisson correction distance measurement of nomore than 0.65, based on alignment of the AAV vp1 amino acid sequence.

The AAV clades which have been described include Clade A (represented byAAV1 and AAV6), Clade B (represented by AAV2) and Clade C (representedby the AAV2-AAV3 hybrid), Clade D (represented by AAV7), Clade E(represented by AAV8), and Clade F (represented by human AAV9). Theseclades are represented by a member of the clade that is a previouslydescribed AAV serotype. Previously described AAV1 and AAV6 are membersof a single clade (Clade A) in which 4 isolates were recovered from 3humans. Previously described AAV3 and AAV5 serotypes are clearlydistinct from one another, but were not detected in the screen describedherein, and have not been included in any of these clades.

Further discussion of AAV clades is provided in G. Gao, et al., J.Virol., 78(12):6381-6388 (June 2004) and International PatentPublication Nos. WO 2004/028817 and WO2005/033321. The latter documentalso provides novel human AAV sequences, which are incorporated byreference herein.

In one embodiment, the libraries used in the method of the inventionexclude AAV5. In another embodiment, the libraries used in the method ofthe invention exclude AAV4. However, in certain embodiments, e.g., wherethe parental AAV is similar to AAV5, it may be desirable to include thissequence in the alignment.

Although a library can be constructed that contains the minimal numberof sequences, efficiency in identifying singletons may be optimized byutilizing a library containing a larger number of sequences. Suitably,the library contains a minimum of four sequences, with at least twoclades being represented. Preferably, the library contains at least twosequences from each of the represented clades. In one embodiment, thelibrary contains more than 100 AAV sequences. In another embodiment, thelibrary contains at least three to 100 AAV sequences. In still anotherembodiment, the library contains at least six to 50 AAV sequences.

Suitable AAVs for use in the functional libraries of the inventioninclude, e.g., AAV1, AAV2, AAV6, AAV7, AAV8, AAV9, and other sequenceswhich have been described [G. Gao, et al, Proc Natl. Acad Sci.,100(10):6081-6086 (May 13, 2003); International Patent Publication Nos.WO 2004/042397 and WO 2005/033321]. One of skill in the art can readilyselect other AAVs, e.g., those isolated using the methods described inInternational Patent Publication No. WO 03/093460 A1 (Nov. 13, 2003) andUS Patent Application Publication No. 2003-0138772 A1 (Jul. 24, 2003).

According to the present invention, the at least three sequences withinthe library are least 85% identical over the full-length of theiraligned capsid sequences.

The term “percent (%) identity” may be readily determined for amino acidsequences, over the full-length of a protein, or a fragment thereof.Suitably, a fragment is at least about 8 amino acids in length, and maybe up to about 700 amino acids. Generally, when referring to “identity”,“homology”, or “similarity” between two different adeno-associatedviruses, “identity”, “homology” or “similarity” is determined inreference to “aligned” sequences. “Aligned” sequences or “alignments”refer to multiple nucleic acid sequences or protein (amino acids)sequences, often containing corrections for missing or additional basesor amino acids as compared to a reference sequence.

Alignments are performed using any of a variety of publicly orcommercially available Multiple Sequence Alignment Programs, Sequencealignment programs are available for amino acid sequences, e.g., the“Clustal X”, “MAP”, “PIMA”, “MSA”, “BLOCKMAKER”, “MEME”, and “Match-Box”programs. Generally, any of these programs are used at default settings,although one of skill in the art can alter these settings as needed.Alternatively, one of skill in the art can utilize another algorithm orcomputer program which provides at least the level of identity oralignment as that provided by the referenced algorithms and programs.See, e.g., J. D. Thomson et al, Nucl. Acids. Res., “A comprehensivecomparison of multiple sequence alignments”, 27(13):2682-2690 (1999).

Multiple sequence alignment programs are also available for nucleic acidsequences. Examples of such programs include, “Clustal W”, “CAP SequenceAssembly”, “MAP”, and “MEME”, which are accessible through Web Serverson the internet. Other sources for such programs are known to those ofskill in the art. Alternatively, Vector NTI utilities are also used.There are also a number of algorithms known in the art that can be usedto measure nucleotide sequence identity, including those contained inthe programs described above. As another example, polynucleotidesequences can be compared using Fasta™, a program in GCG Version 6.1.Fasta™ provides alignments and percent sequence identity of the regionsof the best overlap between the query and search sequences. Forinstance, percent sequence identity between nucleic acid sequences canbe determined using Fasta™ with its default parameters (a word size of 6and the NOPAM factor for the scoring matrix) as provided in GCG Version6.1, herein incorporated by reference.

According to the invention, the sequences of the target or parental AAVcapsid suspected of containing a singleton are compared to the sequencesof the AAV capsids within the library. This comparison is performedusing an alignment of the full-length vp1 protein of the AAV capsid.

A singleton is identified where, for a selected amino acid position whenthe AAV sequences are aligned, all of the AAVs in the library have thesame amino acid residue (i.e., are completely conserved), but theparental AAV has a different amino acid residue.

Typically, when an alignment is prepared based upon the AAV capsid vp1protein, the alignment contains insertions and deletions which are soidentified with respect to a reference AAV sequence (e.g., AAV2) and thenumbering of the amino acid residues is based upon a reference scaleprovided for the alignment. However, any given AAV sequence may havefewer amino acid residues than the reference scale. In the presentinvention, when discussing the parental AAV and the sequences of thereference library, the term “the same position” or the “correspondingposition” refers to the amino acid located at the same residue number ineach of the sequences, with respect to the reference scale for thealigned sequences. However, when taken out of the alignment, each of theAAV vp1 proteins may have these amino acids located at different residuenumbers.

Optionally, the method of the invention can be performed using a nucleicacid alignment and identifying as a singleton a codon which encodes adifferent amino acid (i.e., a non-synonymous codon). Where the nucleicacid sequences of a given codon are not identical in the parental AAV ascompared to the sequences of that codon in the library, but encode thesame amino acid, they are considered synonymous and are not a singleton.

According to the present invention, a parental AAV containing asingleton is altered such that the singleton residue is replaced withthe conserved amino acid residue of the AAVs in the library.

Conveniently, this replacement can be performed by using conventionalsite-directed mutagenesis techniques on the codon for the variable aminoacid. Typically, the site-directed mutagenesis is performed using as fewsteps as required to obtain the desired codon for the conserved aminoacid residue. Such methods are well known to those of skill in the artand can be performed using published methods and/or commerciallyavailable kits [e.g., available from Stratagene and Promega]. Thesite-directed mutagenesis may be performed on the AAV genomic sequence.The AAV sequence may be carried by a vector (e.g., a plasmid backbone)for convenience. Alternatively, one of skill in the art can alter theparental AAV using other techniques know to those of skill in the art.

A parental AAV may have more than one singleton, e.g., two, three, four,five, six or more. However, improvement in function may be observedafter correction of one singleton. In the embodiment where a parentalAAV carries multiple singletons, each singleton may be altered at atime, followed by assessment of the modified AAV for enhancement of thedesired function. Alternatively, multiple singletons may be alteredprior to assessment for enhancement of the desired function.

Even where a parental AAV contains multiple singletons and functionalimprovement is observed altered of a first singleton, it may bedesirable to optimize function by altering the remaining singleton(s).

Typically, a parental AAV which has had one or more singleton(s) alteredaccording to the method of the invention, is assessed for function bypackaging the AAV into an AAV particle. These methods are well known tothose of skill in the art. See, e.g., G. Gao et al, Proc Natl Acad Sci.,cited above; Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor N.Y.

These altered AAVs have novel capsids produced according to the methodof the invention and are assessed for function. Suitable methods forassessing AAV function have been described herein and include, e.g., theability to produce DNAse protected particles, in vitro cell transductionefficiency, and/or in vivo gene transfer. Suitably, the altered AAVs ofthe invention have a sufficient number of singletons altered to increasefunction in one or all of these characteristics, as compared to thefunction of the parent AAV.

II. Novel AAV of the Invention

The invention further provides a method of predicting whether a novelAAV will be functional. The method involves using the singleton methodof the invention and identifying the absence of a singleton in thesequence of the selected AAV, i.e., an AAV which lacks a singleton.

Thus, in one embodiment, the invention provides a method of selecting anAAV for use in producing a vector. This method involves selecting aparental AAV capsid sequence for analysis and identifying the absence ofany singletons in the parental AAV capsid in an alignment comprising theparental AAV capsid sequence and a library of functional AAV capsidsequences. Once the absence of a singleton in a selected AAV capsid isdetermined, the AAV can be used to generate a vector according to knowntechniques.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid, or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or anopen reading frame thereof, or another suitable fragment which is atleast 15 nucleotides in length. Examples of suitable fragments aredescribed herein.

The terms “sequence identity”, “percent sequence identity”, or “percentidentical” in the context of nucleic acid sequences refers to theresidues in the two sequences which are the same when aligned formaximum correspondence. The length of sequence identity comparison maybe over the full-length of the genome, the full-length of a gene codingsequence, or a fragment of at least about 500 to 5000 nucleotides, isdesired. However, identity among smaller fragments, e.g. of at leastabout nine nucleotides, usually at least about 20 to 24 nucleotides, atleast about 28 to 32 nucleotides, at least about 36 or more nucleotides,may also be desired.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid (or its complementary strand), there is aminoacid sequence identity in at least about 95 to about 99% of the alignedsequences and in certain embodiments, about 97% of the alignedsequences. Preferably, the homology is over full-length sequence, or aprotein thereof, e.g., a cap protein, a rep protein, or a fragmentthereof which is at least 8 amino acids, or more desirably, at least 15amino acids in length. Examples of suitable fragments are describedherein.

By the term “highly conserved” is meant at least 80% identity,preferably at least 90% identity, and more preferably, over 97%identity. Identity is readily determined by one of skill in the art byresort to algorithms and computer programs known by those of skill inthe art.

The term “serotype” is a distinction with respect to an AAV having acapsid which is serologically distinct from other AAV serotypes.Serologic distinctiveness is determined on the basis of the lack ofcross-reactivity between antibodies to the AAV as compared to other AAV.Cross-reactivity is typically measured in a neutralizing antibody assay.For this assay polyclonal serum is generated against a specific AAV in arabbit or other suitable animal model using the adeno-associatedviruses. In this assay, the serum generated against a specific AAV isthen tested in its ability to neutralize either the same (homologous) ora heterologous AAV. The dilution that achieves 50% neutralization isconsidered the neutralizing antibody titer. If for two AAVs the quotientof the heterologous titer divided by the homologous titer is lower than16 in a reciprocal manner, those two vectors are considered as the sameserotype. Conversely, if the ratio of the heterologous titer over thehomologous titer is 16 or more in a reciprocal manner the two AAVs areconsidered distinct serotypes.

In a further embodiment, the invention provides AAV having novelcapsids, including rh.20, rh.32/33, rh.39, rh.46, rh.73, and rh.74. Thesequences of rh.20 have the amino acid sequence of SEQ ID NO:1, or asequence 95 to 99% identical over the full-length of SEQ ID NO:1. Thecapsid of rh.32/33 has an amino acid sequence of SEQ ID NO:2, orsequences 95% to 99% identical thereto over the full-length of SEQ IDNO:2. The capsid of rh.39 has an amino acid sequence of SEQ ID NO:3, orsequences 95% to 99% identical thereto over the full-length of SEQ IDNO:3. The capsid of rh.46 has an amino acid sequence of SEQ ID NO:4, orsequences 95% to 99% identical thereto over the full-length of SEQ IDNO:4. The capsid of rh.73 has an amino acid sequence of SEQ ID NO:5, orsequences 95% to 99% identical thereto over the full-length of SEQ IDNO:5. The capsid of rh.74 has an amino acid sequence of SEQ ID NO:6, orsequences 95% to 99% identical thereto over the full-length of SEQ IDNO:6. Preferably, the sequence identity of these novel AAV capsids issuch that it lacks any singletons. The sequences of the novel AAV areprovided in the Sequence Listing.

In still another embodiment, the novel AAV sequences of the inventioninclude the singleton-corrected AAV capsid proteins and the sequencesencoding these capsid proteins. Examples of suitable singleton-correctAAV sequences include, AAV6.1, AAV6.2, AAV6.1.2, rh.8R, rh.48.1,rh.48.2, rh.48.1.2, hu.44R1, hu.44R2 hu.44R3, hu.29R, ch.5R1, rh.67,rh.54, hu.48R1, hu.48R2, and hu.48R3. For example, thesingleton-corrected AAV6, including AAV6.1, AAV6.2 and AAV6.12 haveshown significant functional improvement over the parental AAV6sequence.

Particularly desirable proteins include the AAV capsid proteins, whichare encoded by the nucleotide sequences identified above. The AAV capsidis composed of three proteins, vp1, vp2 and vp3, which are alternativesplice variants. Other desirable fragments of the capsid protein includethe constant and variable regions, located between hypervariable regions(HVR). Other desirable fragments of the capsid protein include the HVRthemselves.

An algorithm developed to determine areas of sequence divergence in AAV2has yielded 12 hypervariable regions (HVR) of which 5 overlap or arepart of the four previously described variable regions. [Chiorini et al,J. Virol, 73:1309-19 (1999); Rutledge et al, J. Virol., 72:309-319]Using this algorithm and/or the alignment techniques described herein,the HVR of the novel AAV serotypes are determined. For example, the HVRare located as follows: HVR1, aa 146-152; HVR2, aa 182-186; HVR3, aa262-264; HVR4, aa 381-383; HVR5, aa 450-474; HVR6, aa 490-495; HVR7, aa500-504; HVR8, aa 514-522; HVR9, aa 534-555; HVR10, aa 581-594; HVR11,aa 658-667; and HVR12, aa 705-719 [the numbering system is based on analignment which uses the AAV2 vp1 as a point of reference]. Using thealignment provided herein performed using the Clustal X program atdefault settings, or using other commercially or publicly availablealignment programs at default settings such as are described herein, oneof skill in the art can readily determine corresponding fragments of thenovel AAV capsids of the invention.

Suitably, fragments are at least 8 amino acids in length. However,fragments of other desired lengths may be readily utilized. Suchfragments may be produced recombinantly or by other suitable means,e.g., by chemical synthesis.

The invention further provides other AAV sequences which are identifiedusing the sequence information provided herein. For example, given thesequences provided herein, infectious may be isolated using genomewalking technology (Siebert et al., 1995, Nucleic Acid Research,23:1087-1088, Friezner-Degen et al., 1986, J. Biol. Chem. 261:6972-6985,BD Biosciences Clontech, Palo Alto, Calif.). Genome walking isparticularly well suited for identifying and isolating the sequencesadjacent to the novel sequences identified according to the method ofthe invention. This technique is also useful for isolating invertedterminal repeat (ITRs) of the novel AAV, based upon the novel AAV capsidand rep sequences provided herein.

The novel AAV amino acid sequences, peptides and proteins may beexpressed from AAV nucleic acid sequences of the invention.Additionally, these amino acid sequences, peptides and proteins can begenerated by other methods known in the art, including, e.g., bychemical synthesis, by other synthetic techniques, or by other methods.The sequences of any of the AAV capsids provided herein can be readilygenerated using a variety of techniques.

Suitable production techniques are well known to those of skill in theart. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively,peptides can also be synthesized by the well-known solid phase peptidesynthesis methods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962);Stewart and Young, Solid Phase Peptide Synthesis (Freeman, SanFrancisco, 1969) pp. 27-62). These and other suitable production methodsare within the knowledge of those of skill in the art and are not alimitation of the present invention.

The sequences, proteins, and fragments of the invention may be producedby any suitable means, including recombinant production, chemicalsynthesis, or other synthetic means. Such production methods are withinthe knowledge of those of skill in the art and are not a limitation ofthe present invention.

III. Production of rAAV with Novel AAV Capsids

The invention encompasses novel AAV capsid sequences generated bymutation following use of the method of the invention for identifyingsingletons. The invention further encompasses the novel AAV rh.20,rh.32/33, rh.39, rh.46, rh.73, and rh.74 capsid sequences [SEQ ID Nos:1-6].

In another aspect, the present invention provides molecules that utilizethe novel AAV sequences of the invention, including fragments thereof,for production of viral vectors useful in delivery of a heterologousgene or other nucleic acid sequences to a target cell.

The molecules of the invention which contain AAV sequences include anygenetic element (vector) which may be delivered to a host cell, e.g.,naked DNA, a plasmid, phage, transposon, cosmid, episome, a protein in anon-viral delivery vehicle (e.g., a lipid-based carrier), virus, etc.,which transfers the sequences carried thereon.

The selected vector may be delivered by any suitable method, includingtransfection, electroporation, liposome delivery, membrane fusiontechniques, high velocity DNA-coated pellets, viral infection andprotoplast fusion. The methods used to construct any embodiment of thisinvention are known to those with skill in nucleic acid manipulation andinclude genetic engineering, recombinant engineering, and synthetictechniques. See, e.g., Sambrook et al, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.

In one embodiment, the vectors of the invention contain, inter alia,sequences encoding an AAV capsid of the invention or a fragment thereof.In another embodiment, the vectors of the invention contain, at aminimum, sequences encoding an AAV rep protein or a fragment thereof.Optionally, vectors of the invention may contain both AAV cap and repproteins. In vectors in which both AAV rep and cap are provided, the AAVrep and AAV cap sequences can originate from an AAV of the same clade.Alternatively, the present invention provides vectors in which the repsequences are from an AAV source which differs from that which isproviding the cap sequences. In one embodiment, the rep and capsequences are expressed from separate sources (e.g., separate vectors,or a host cell and a vector). In another embodiment, these rep sequencesare fused in frame to cap sequences of a different AAV source to form achimeric AAV vector. Optionally, the vectors of the invention arevectors packaged in an AAV capsid of the invention. These vectors andother vectors described herein can further contain a minigene comprisinga selected transgene which is flanked by AAV 5′ ITR and AAV 3′ ITR.

Thus, in one embodiment, the vectors described herein contain nucleicacid sequences encoding an intact AAV capsid which may be from a singleAAV sequence. Alternatively, these vectors contain sequences encodingartificial capsids which contain one or more fragments of thesingleton-corrected AAV capsid fused to heterologous AAV or non-AAVcapsid proteins (or fragments thereof). These artificial capsid proteinsare selected from non-contiguous portions of the singleton-correctedcapsid or from capsids of other AAVs. These modifications may be toincrease expression, yield, and/or to improve purification in theselected expression systems, or for another desired purpose (e.g., tochange tropism or alter neutralizing antibody epitopes).

The vectors described herein, e.g., a plasmid, are useful for a varietyof purposes, but are particularly well suited for use in production of arAAV containing a capsid comprising AAV sequences or a fragment thereof.These vectors, including rAAV, their elements, construction, and usesare described in detail herein.

In one aspect, the invention provides a method of generating arecombinant adeno-associated virus (AAV) having a novel AAV capsid ofthe invention. Such a method involves culturing a host cell whichcontains a nucleic acid sequence encoding a novel AAV capsid protein ofthe invention, or fragment thereof, as defined herein; a functional repgene; a minigene composed of, at a minimum, AAV inverted terminalrepeats (ITRs) and a transgene; and sufficient helper functions topermit packaging of the minigene into the AAV capsid protein.

The components required to be cultured in the host cell to package anAAV minigene in an AAV capsid may be provided to the host cell in trans.Alternatively, one or more of the required components (e.g., minigene,rep sequences, cap sequences, and/or helper functions) may be providedby a stable host cell which has been engineered to contain one or moreof the required components using methods known to those of skill in theart. Most suitably, such a stable host cell will contain the requiredcomponent(s) under the control of an inducible promoter. However, therequired component(s) may be under the control of a constitutivepromoter. Examples of suitable inducible and constitutive promoters areprovided herein, in the discussion of regulatory elements suitable foruse with the transgene. In still another alternative, a selected stablehost cell may contain selected component(s) under the control of aconstitutive promoter and other selected component(s) under the controlof one or more inducible promoters. For example, a stable host cell maybe generated which is derived from 293 cells (which contain E1 helperfunctions under the control of a constitutive promoter), but whichcontains the rep and/or cap proteins under the control of induciblepromoters. Still other stable host cells may be generated by one ofskill in the art.

The minigene, rep sequences, cap sequences, and helper functionsrequired for producing the rAAV of the invention may be delivered to thepackaging host cell in the form of any genetic element which transferthe sequences carried thereon. The selected genetic element may bedelivered by any suitable method, including those described herein. Themethods used to construct any embodiment of this invention are known tothose with skill in nucleic acid manipulation and include geneticengineering, recombinant engineering, and synthetic techniques. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. Similarly, methods ofgenerating rAAV virions are well known and the selection of a suitablemethod is not a limitation on the present invention. See, e.g., K.Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

Unless otherwise specified, the AAV ITRs, and other selected AAVcomponents described herein, may be readily selected from among any AAV,including, without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV9 and one of the other novel AAV sequences of the invention. TheseITRs or other AAV components may be readily isolated using techniquesavailable to those of skill in the art from an AAV sequence. Such AAVmay be isolated or obtained from academic, commercial, or public sources(e.g., the American Type Culture Collection, Manassas, Va.).Alternatively, the AAV sequences may be obtained through synthetic orother suitable means by reference to published sequences such as areavailable in the literature or in databases such as, e.g., GenBank®,PubMed®, or the like.

A. The Minigene

The minigene is composed of, at a minimum, a transgene and itsregulatory sequences, and 5′ and 3′ AAV inverted terminal repeats(ITRs). In one desirable embodiment, the ITRs of AAV serotype 2 areused. However, ITRs from other suitable sources may be selected. It isthis minigene that is packaged into a capsid protein and delivered to aselected host cell.

1. The Transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),enhanced GFP (EGFP), chloramphenicol acetyltransferase (CAT),luciferase, membrane bound proteins including, for example, CD2, CD4,CD8, the influenza hemagglutinin protein, and others well known in theart, to which high affinity antibodies directed thereto exist or can beproduced by conventional means, and fusion proteins comprising amembrane bound protein appropriately fused to an antigen tag domainfrom, among others, hemagglutinin or Myc.

These coding sequences, when associated with regulatory elements whichdrive their expression, provide signals detectable by conventionalmeans, including enzymatic, radiographic, colorimetric, fluorescence orother spectrographic assays, fluorescent activating cell sorting assaysand immunological assays, including enzyme linked immunosorbent assay(ELISA), radioimmunoassay (RIA) and immunohistochemistry. For example,where the marker sequence is the LacZ gene, the presence of the vectorcarrying the signal is detected by assays for beta-galactosidaseactivity. Where the transgene is green fluorescent protein orluciferase, the vector carrying the signal may be measured visually bycolor or light production in a luminometer.

However, desirably, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, dominant negative mutants, or catalytic RNAs.Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalyticRNAs, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNAs.One example of a useful RNA sequence is a sequence which inhibits orextinguishes expression of a targeted nucleic acid sequence in thetreated animal. Typically, suitable target sequences include oncologictargets and viral diseases. See, for examples of such targets theoncologic targets and viruses identified below in the section relatingto immunogens.

The transgene may be used to correct or ameliorate gene deficiencies,which may include deficiencies in which normal genes are expressed atless than normal levels or deficiencies in which the functional geneproduct is not expressed. Alternatively, the transgene may provide aproduct to a cell which is not natively expressed in the cell type or inthe host. A preferred type of transgene sequence encodes a therapeuticprotein or polypeptide which is expressed in a host cell. The inventionfurther includes using multiple transgenes. In certain situations, adifferent transgene may be used to encode each subunit of a protein, orto encode different peptides or proteins. This is desirable when thesize of the DNA encoding the protein subunit is large, e.g. for animmunoglobulin, the platelet-derived growth factor, or a dystrophinprotein. In order for the cell to produce the multi-subunit protein, acell is infected with the recombinant virus containing each of thedifferent subunits. Alternatively, different subunits of a protein maybe encoded by the same transgene. In this case, a single transgeneincludes the DNA encoding each of the subunits, with the DNA for eachsubunit separated by an internal ribozyme entry site (IRES). This isdesirable when the size of the DNA encoding each of the subunits issmall, e.g., the total size of the DNA encoding the subunits and theIRES is less than five kilobases. As an alternative to an IRES, the DNAmay be separated by sequences encoding a 2A peptide, which self-cleavesin a post-translational event. See, e.g., M. L. Donnelly, et al, J. Gen.Virol., 78(Pt 1):13-21 (January 1997); Furler, S., et al, Gene Ther.,8(11):864-873 (June 2001); Klump H., et al., Gene Ther., 8(10):811-817(May 2001). This 2A peptide is significantly smaller than an IRES,making it well suited for use when space is a limiting factor. Moreoften, when the transgene is large, consists of multi-subunits, or twotransgenes are co-delivered, rAAV carrying the desired transgene(s) orsubunits are co-administered to allow them to concatamerize in vivo toform a single vector genome. In such an embodiment, a first AAV maycarry an expression cassette which expresses a single transgene and asecond AAV may carry an expression cassette which expresses a differenttransgene for co-expression in the host cell. However, the selectedtransgene may encode any biologically active product or other product,e.g., a product desirable for study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis invention.

2. Regulatory Elements

In addition to the major elements identified above for the minigene, thevector also includes conventional control elements which are operablylinked to the transgene in a manner which permits its transcription,translation and/or expression in a cell transfected with the plasmidvector or infected with the virus produced by the invention. As usedherein, “operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], theSV40 promoter, the dihydrofolate reductase promoter, the β-actinpromoter, the phosphoglycerol kinase (PGK) promoter, and the EF1promoter [Invitrogen]. Inducible promoters allow regulation of geneexpression and can be regulated by exogenously supplied compounds,environmental factors such as temperature, or the presence of a specificphysiological state, e.g., acute phase, a particular differentiationstate of the cell, or in replicating cells only. Inducible promoters andinducible systems are available from a variety of commercial sources,including, without limitation, Invitrogen, Clontech and Ariad. Manyother systems have been described and can be readily selected by one ofskill in the art. Examples of inducible promoters regulated byexogenously supplied compounds, include, the zinc-inducible sheepmetallothionine (MT) promoter, the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter, the T7 polymerase promoter system[International Patent Publication No. WO 98/10088]; the ecdysone insectpromoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)],the tetracycline-repressible system [Gossen et al, Proc. Natl. Acad.Sci. USA, 89:5547-5551 (1992)], the tetracycline-inducible system[Gossen et al, Science, 268:1766-1769 (1995), see also Harvey et al,Curr. Opin. Chem. Biol., 2:512-518 (1998)], the RU486-inducible system[Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al, GeneTher., 4:432-441 (1997)] and the rapamycin-inducible system [Magari etal, J. Clin. Invest., 100:2865-2872 (1997)]. Other types of induciblepromoters which may be useful in this context are those which areregulated by a specific physiological state, e.g., temperature, acutephase, a particular differentiation state of the cell, or in replicatingcells only.

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a gene operably linked to atissue-specific promoter. For instance, if expression in skeletal muscleis desired, a promoter active in muscle should be used. These includethe promoters from genes encoding skeletal β-actin, myosin light chain2A, dystrophin, muscle creatine kinase, as well as synthetic musclepromoters with activities higher than naturally-occurring promoters (seeLi et al., Nat. Biotech., 17:241-245 (1999)). Examples of promoters thatare tissue-specific are known for liver (albumin, Miyatake et al., J.Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig etal., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot etal., Hum. Gene Ther., 7:1503-14 (1996)), bone osteocalcin (Stein et al.,Mol. Biol. Rep., 24:185-96 (1997)); bone sialoprotein (Chen et al., J.Bone Miner. Res., 11:654-64 (1996)), lymphocytes (CD2, Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptorchain), neuronal such as neuron-specific enolase (NSE) promoter(Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)),neurofilament light-chain gene (Piccioli et al., Proc. Natl. Acad. Sci.USA, 88:5611-5 (1991)), and the neuron-specific vgf gene (Piccioli etal., Neuron, 15:373-84 (1995)), among others.

Optionally, plasmids carrying therapeutically useful transgenes may alsoinclude selectable markers or reporter genes may include sequencesencoding geneticin, hygromycin or puromycin resistance, among others.Such selectable reporters or marker genes (preferably located outsidethe viral genome to be rescued by the method of the invention) can beused to signal the presence of the plasmids in bacterial cells, such asampicillin resistance. Other components of the plasmid may include anorigin of replication. Selection of these and other promoters and vectorelements are conventional and many such sequences are available [see,e.g., Sambrook et al, and references cited therein].

The combination of the transgene, promoter/enhancer, and 5′ and 3′ AAVITRs is referred to as a “minigene” for ease of reference herein.Provided with the teachings of this invention, the design of such aminigene can be made by resort to conventional techniques.

3. Delivery of the Minigene to a Packaging Host Cell

The minigene can be carried on any suitable vector, e.g., a plasmid,which is delivered to a host cell. The plasmids useful in this inventionmay be engineered such that they are suitable for replication and,optionally, integration in prokaryotic cells, mammalian cells, or both.These plasmids (or other vectors carrying the 5′ AAV ITR-heterologousmolecule-3′ AAV ITR) contain sequences permitting replication of theminigene in eukaryotes and/or prokaryotes and selection markers forthese systems. Selectable markers or reporter genes may includesequences encoding geneticin, hygromycin or puromycin resistance, amongothers. The plasmids may also contain certain selectable reporters ormarker genes that can be used to signal the presence of the vector inbacterial cells, such as ampicillin resistance. Other components of theplasmid may include an origin of replication and an amplicon, such asthe amplicon system employing the Epstein Barr virus nuclear antigen.This amplicon system, or other similar amplicon components permit highcopy episomal replication in the cells. Preferably, the moleculecarrying the minigene is transfected into the cell, where it may existtransiently. Alternatively, the minigene (carrying the 5′ AAVITR-heterologous molecule-3′ ITR) may be stably integrated into thegenome of the host cell, either chromosomally or as an episome. Incertain embodiments, the minigene may be present in multiple copies,optionally in head-to-head, head-to-tail, or tail-to-tail concatamers.Suitable transfection techniques are known and may readily be utilizedto deliver the minigene to the host cell.

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, about 10 μg to about 50 μg DNA to about 1×10⁴ cells toabout 1×10¹³ cells, or about 1×10⁵ cells. However, the relative amountsof vector DNA to host cells may be adjusted by one of ordinary skill inthe art, who may take into consideration such factors as the selectedvector, the delivery method and the host cells selected.

B. Rep and Cap Sequences

In addition to the minigene, the host cell contains the sequences whichdrive expression of a novel AAV capsid protein of the invention (or acapsid protein comprising a fragment thereof) in the host cell and repsequences of the same source as the source of the AAV ITRs found in theminigene, or a cross-complementing source. The AAV cap and rep sequencesmay be independently obtained from an AAV source as described above andmay be introduced into the host cell in any manner known to one in theart as described above. Additionally, when pseudotyping an AAV vector,the sequences encoding each of the essential rep proteins may besupplied by different AAV sources (e.g., AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9). For example, the rep78168 sequences may be fromAAV2, whereas the repS2/40 sequences may be from AAV8.

In one embodiment, the host cell stably contains the capsid proteinunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the capsid protein is expressedunder the control of an inducible promoter. In another embodiment, thecapsid protein is supplied to the host cell in trans. When delivered tothe host cell in trans, the capsid protein may be delivered via aplasmid which contains the sequences necessary to direct expression ofthe selected capsid protein in the host cell. Most desirably, whendelivered to the host cell in trans, the plasmid carrying the capsidprotein also carries other sequences required for packaging the rAAV,e.g., the rep sequences.

In another embodiment, the host cell stably contains the rep sequencesunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the essential rep proteins areexpressed under the control of an inducible promoter. In anotherembodiment, the rep proteins are supplied to the host cell in trans.When delivered to the host cell in trans, the rep proteins may bedelivered via a plasmid which contains the sequences necessary to directexpression of the selected rep proteins in the host cell. Mostdesirably, when delivered to the host cell in trans, the plasmidcarrying the capsid protein also carries other sequences required forpackaging the rAAV, e.g., the rep and cap sequences.

Thus, in one embodiment, the rep and cap sequences may be transfectedinto the host cell on a single nucleic acid molecule and exist stably inthe cell as an episome. In another embodiment, the rep and cap sequencesare stably integrated into the chromosome of the cell. Anotherembodiment has the rep and cap sequences transiently expressed in thehost cell. For example, a useful nucleic acid molecule for suchtransfection comprises, from 5′ to 3′, a promoter, an optional spacerinterposed between the promoter and the start site of the rep genesequence, an AAV rep gene sequence, and an AAV cap gene sequence.

Optionally, the rep and/or cap sequences may be supplied on a vectorthat contains other DNA sequences that are to be introduced into thehost cells. For instance, the vector may contain the rAAV constructcomprising the minigene. The vector may comprise one or more of thegenes encoding the helper functions, e.g., the adenoviral proteins E1,E2a, and E4 ORF6, and the gene for VAI RNA.

Preferably, the promoter used in this construct may be any of theconstitutive, inducible or native promoters known to one of skill in theart or as discussed above. In one embodiment, an AAV P5 promotersequence is employed. The selection of the AAV to provide any of thesesequences does not limit the invention.

In another preferred embodiment, the promoter for rep is an induciblepromoter, such as are discussed above in connection with the transgeneregulatory elements. One preferred promoter for rep expression is the T7promoter. The vector comprising the rep gene regulated by the T7promoter and the cap gene, is transfected or transformed into a cellwhich either constitutively or inducibly expresses the T7 polymerase.See International Patent Publication No. WO 98/10088, published Mar. 12,1998.

The spacer is an optional element in the design of the vector. Thespacer is a DNA sequence interposed between the promoter and the repgene ATG start site. The spacer may have any desired design; that is, itmay be a random sequence of nucleotides, or alternatively, it may encodea gene product, such as a marker gene. The spacer may contain geneswhich typically incorporate start/stop and polyA sites. The spacer maybe a non-coding DNA sequence from a prokaryote or eukaryote, arepetitive non-coding sequence, a coding sequence withouttranscriptional controls or a coding sequence with transcriptionalcontrols. Two exemplary sources of spacer sequences are the phage laddersequences or yeast ladder sequences, which are available commercially,e.g., from Gibco or Invitrogen, among others. The spacer may be of anysize sufficient to reduce expression of the rep78 and rep68 geneproducts, leaving the rep52, rep40 and cap gene products expressed atnormal levels. The length of the spacer may therefore range from about10 bp to about 10.0 kbp, preferably in the range of about 100 bp toabout 8.0 kbp. To reduce the possibility of recombination, the spacer ispreferably less than 2 kbp in length; however, the invention is not solimited.

Although the molecule(s) providing rep and cap may exist in the hostcell transiently (i.e., through transfection), it is preferred that oneor both of the rep and cap proteins and the promoter(s) controllingtheir expression be stably expressed in the host cell, e.g., as anepisome or by integration into the chromosome of the host cell. Themethods employed for constructing embodiments of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above. While thisspecification provides illustrative examples of specific constructs,using the information provided herein, one of skill in the art mayselect and design other suitable constructs, using a choice of spacers,P5 promoters, and other elements, including at least one translationalstart and stop signal, and the optional addition of polyadenylationsites.

In another embodiment of this invention, the rep or cap protein may beprovided stably by a host cell.

C. The Helper Functions

The packaging host cell also requires helper functions in order topackage the rAAV of the invention. Optionally, these functions may besupplied by a herpesvirus. Most desirably, the necessary helperfunctions are each provided from a human or non-human primate adenovirussource, such as those described above and/or are available from avariety of sources, including the American Type Culture Collection(ATCC), Manassas, Va. (US). In one currently preferred embodiment, thehost cell is provided with and/or contains an E1a gene product, an E1bgene product, an E2a gene product, and/or an E4 ORF6 gene product. Thehost cell may contain other adenoviral genes such as VAI RNA, but thesegenes are not required. In a preferred embodiment, no other adenovirusgenes or gene functions are present in the host cell.

By “adenoviral DNA which expresses the E1a gene product”, it is meantany adenovirus sequence encoding E1a or any functional E1a portion.Adenoviral DNA which expresses the E2a gene product and adenoviral DNAwhich expresses the E4 ORF6 gene products are defined similarly. Alsoincluded are any alleles or other modifications of the adenoviral geneor functional portion thereof. Such modifications may be deliberatelyintroduced by resort to conventional genetic engineering or mutagenictechniques to enhance the adenoviral function in some manner, as well asnaturally occurring allelic variants thereof. Such modifications andmethods for manipulating DNA to achieve these adenovirus gene functionsare known to those of skill in the art.

The adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products, as well asany other desired helper functions, can be provided using any means thatallows their expression in a cell. Each of the sequences encoding theseproducts may be on a separate vector, or one or more genes may be on thesame vector. The vector may be any vector known in the art or disclosedabove, including plasmids, cosmids and viruses. Introduction into thehost cell of the vector may be achieved by any means known in the art oras disclosed above, including transfection, infection, electroporation,liposome delivery, membrane fusion techniques, high velocity DNA-coatedpellets, viral infection and protoplast fusion, among others. One ormore of the adenoviral genes may be stably integrated into the genome ofthe host cell, stably expressed as episomes, or expressed transiently.The gene products may all be expressed transiently, on an episome orstably integrated, or some of the gene products may be expressed stablywhile others are expressed transiently. Furthermore, the promoters foreach of the adenoviral genes may be selected independently from aconstitutive promoter, an inducible promoter or a native adenoviralpromoter. The promoters may be regulated by a specific physiologicalstate of the organism or cell (i.e., by the differentiation state or inreplicating or quiescent cells) or by other means, e.g., by exogenouslyadded factors.

D. Host Cells And Packaging Cell Lines

The host cell itself may be selected from any biological organism,including prokaryotic (e.g., bacterial) cells, and eukaryotic cells,including, insect cells, yeast cells and mammalian cells. Particularlydesirable host cells are selected from among any mammalian species,including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2,BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293cells (which express functional adenoviral E1), Saos, C2C12, L cells,HT1080, HepG2 and primary fibroblast, hepatocyte and myoblast cellsderived from mammals including human, monkey, mouse, rat, rabbit, andhamster. The selection of the mammalian species providing the cells isnot a limitation of this invention; nor is the type of mammalian cell,i.e., fibroblast, hepatocyte, tumor cell, etc. The requirements for thecell used is that it not carry any adenovirus gene other than E1, E2aand/or E4 ORF6; it not contain any other virus gene which could resultin homologous recombination of a contaminating virus during theproduction of rAAV; and it is capable of infection or transfection ofDNA and expression of the transfected DNA. In a preferred embodiment,the host cell is one that has rep and cap stably transfected in thecell.

One host cell useful in the present invention is a host cell stablytransformed with the sequences encoding rep and cap, and which istransfected with the adenovirus E1, E2a, and E4ORF6 DNA and a constructcarrying the minigene as described above. Stable rep and/or capexpressing cell lines, such as B-50 (International Patent ApplicationPublication No. WO 99/15685), or those described in U.S. Pat. No.5,658,785, may also be similarly employed. Another desirable host cellcontains the minimum adenoviral DNA which is sufficient to express E4ORF6. Yet other cell lines can be constructed using the novelsingleton-corrected AAV cap sequences of the invention.

The preparation of a host cell according to this invention involvestechniques such as assembly of selected DNA sequences. This assembly maybe accomplished utilizing conventional techniques. Such techniquesinclude cDNA and genomic cloning, which are well known and are describedin Sambrook et al., cited above, use of overlapping oligonucleotidesequences of the adenovirus and AAV genomes, combined with polymerasechain reaction, synthetic methods, and any other suitable methods whichprovide the desired nucleotide sequence.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation, and/or infection by hybridadenovirus/AAV vectors into cell lines such as the human embryonickidney cell line HEK 293 (a human kidney cell line containing functionaladenovirus E1 genes which provides trans-acting E1 proteins).

One of skill in the art will readily understand that the novel AAVsequences of the invention can be readily adapted for use in these andother viral vector systems for in vitro, ex vivo or in vivo genedelivery. Similarly, one of skill in the art can readily select otherfragments of the AAV genome of the invention for use in a variety ofrAAV and non-rAAV vector systems. Such vectors systems may include,e.g., lentiviruses, retroviruses, poxviruses, vaccinia viruses, andadenoviral systems, among others. Selection of these vector systems isnot a limitation of the present invention.

Thus, the invention further provides vectors generated using the nucleicacid and amino acid sequences of the novel AAV of the invention. Suchvectors are useful for a variety of purposes, including for delivery oftherapeutic molecules and for use in vaccine regimens. Particularlydesirable for delivery of therapeutic molecules are recombinant AAVcontaining capsids of the novel AAV of the invention. These, or othervector constructs containing novel AAV sequences of the invention may beused in vaccine regimens, e.g., for co-delivery of a cytokine, or fordelivery of the immunogen itself.

IV. Recombinant Viruses and Uses Therefor

Using the techniques described herein, one of skill in the art cangenerate a rAAV having a capsid of an AAV of the invention or having acapsid containing one or more fragments of an AAV of the invention. Inone embodiment, a full-length capsid from a singleton-corrected AAV canbe utilized.

A. Delivery of Viruses

In another aspect, the present invention provides a method for deliveryof a transgene to a host which involves transfecting or infecting aselected host cell with a recombinant viral vector generated with thesingleton-corrected AAV (or functional fragments thereof) of theinvention. Methods for delivery are well known to those of skill in theart and are not a limitation of the present invention.

In one desirable embodiment, the invention provides a method forAAV-mediated delivery of a transgene to a host. This method involvestransfecting or infecting a selected host cell with a recombinant viralvector containing a selected transgene under the control of sequencesthat direct expression thereof and the modified capsid proteins of thecapsids.

Optionally, a sample from the host may be first assayed for the presenceof antibodies to a selected AAV source (e.g., a serotype). A variety ofassay formats for detecting neutralizing antibodies are well known tothose of skill in the art. The selection of such an assay is not alimitation of the present invention. See, e.g., Fisher et al, NatureMed., 3(3):306-312 (March 1997) and W. C. Manning et al, Human GeneTherapy, 9:477-485 (Mar. 1, 1998). The results of this assay may be usedto determine which AAV vector containing capsid proteins of a particularsource are preferred for delivery, e.g., by the absence of neutralizingantibodies specific for that capsid source.

In one aspect of this method, the delivery of vector with AAV capsidproteins of the invention may precede or follow delivery of a gene via avector with a different AAV capsid protein. Thus, gene delivery via rAAVvectors may be used for repeat gene delivery to a selected host cell.Desirably, subsequently administered rAAV vectors carry the sametransgene as the first rAAV vector, but the subsequently administeredvectors contain capsid proteins of sources (and preferably, differentserotypes) which differ from the first vector. For example, if a firstvector has a singleton-corrected capsid proteins, subsequentlyadministered vectors may have capsid proteins selected from among theother AAV, optionally, from another serotype or from another clade.

Optionally, multiple rAAV vectors can be used to deliver largetransgenes or multiple transgenes by co-administration of rAAV vectorsconcatamerize in vivo to form a single vector genome. In such anembodiment, a first AAV may carry an expression cassette which expressesa single transgene (or a subunit thereof) and a second AAV may carry anexpression cassette which expresses a second transgene (or a different,subunit) for co-expression in the host cell. A first AAV may carry anexpression cassette which is a first piece of a polycistronic construct(e.g., a promoter and transgene, or subunit) and a second AAV may carryan expression cassette which is a second piece of a polycistronicconstruct (e.g., transgene or subunit and a polyA sequence). These twopieces of a polycistronic construct concatamerize in vivo to form asingle vector genome that co-expresses the transgenes delivered by thefirst and second AAV. In such embodiments, the rAAV vector carrying thefirst expression cassette and the rAAV vector carrying the secondexpression cassette can be delivered in a single pharmaceuticalcomposition. In other embodiments, the two or more rAAV vectors aredelivered as separate pharmaceutical compositions which can beadministered substantially simultaneously, or shortly before or afterone another.

The above-described recombinant vectors may be delivered to host cellsaccording to published methods. The rAAV, preferably suspended in aphysiologically compatible carrier, may be administered to a human ornon-human mammalian patient. Suitable carriers may be readily selectedby one of skill in the art in view of the indication for which thetransfer virus is directed. For example, one suitable carrier includessaline, which may be formulated with a variety of buffering solutions(e.g., phosphate buffered saline). Other exemplary carriers includesterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran,agar, pectin, peanut oil, sesame oil, and water. The selection of thecarrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The vectors are administered in sufficient amounts to transfect thecells and to provide sufficient levels of gene transfer and expressionto provide a therapeutic benefit without undue adverse effects, or withmedically acceptable physiological effects, which can be determined bythose skilled in the medical arts. Conventional and pharmaceuticallyacceptable routes of administration include, but are not limited to,direct delivery to a desired organ (e.g., the liver (optionally via thehepatic artery) or lung), oral, inhalation, intranasal, intratracheal,intraarterial, intraocular, intravenous, intramuscular, subcutaneous,intradermal, and other parental routes of administration. Routes ofadministration may be combined, if desired.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectivehuman dosage of the viral vector is generally in the range of from about0.1 mL to about 100 mL of solution containing concentrations of fromabout 1×10⁹ to 1×10¹⁶ genomes virus vector. A preferred human dosage fordelivery to large organs (e.g., liver, muscle, heart and lung) may beabout 5×10¹⁰ to 5×10¹³ AAV genomes per 1 kg, at a volume of about 1 to100 mL. A preferred dosage for delivery to eye is about 5×10⁹ to 5×10¹²genome copies, at a volume of about 0.1 mL to 1 mL. The dosage will beadjusted to balance the therapeutic benefit against any side effects andsuch dosages may vary depending upon the therapeutic application forwhich the recombinant vector is employed. The levels of expression ofthe transgene can be monitored to determine the frequency of dosageresulting in viral vectors, preferably AAV vectors containing theminigene. Optionally, dosage regimens similar to those described fortherapeutic purposes may be utilized for immunization using thecompositions of the invention.

Examples of therapeutic products and immunogenic products for deliveryby the AAV-containing vectors of the invention are provided below. Thesevectors may be used for a variety of therapeutic or vaccinal regimens,as described herein. Additionally, these vectors may be delivered incombination with one or more other vectors or active ingredients in adesired therapeutic and/or vaccinal regimen.

B. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), platelet-derived growth factor (PDGF), insulin growth factors Iand II (IGF-I and IGF-II), any one of the transforming growth factor αsuperfamily, including TGFα, activins, inhibins, or any of the bonemorphogenic proteins (BMP) BMPs 1-15, any one of theheregulin/neuregulin/ARIA/neu differentiation factor (NDF) family ofgrowth factors, nerve growth factor (NGF), brain-derived neurotrophicfactor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophicfactor (CNTF), glial cell line derived neurotrophic factor (GDNF),neurturin, agrin, any one of the family of semaphorins/collapsins,netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin,sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors α and β,interferons α, β, and γ, stem cell factor, flk-2/flt3 ligand. Geneproducts produced by the immune system are also useful in the invention.These include, without limitations, immunoglobulins IgG, IgM, IgA, IgDand IgE, chimeric immunoglobulins, humanized antibodies, single chainantibodies, T cell receptors, chimeric T cell receptors, single chain Tcell receptors, class I and class II MHC molecules, as well asengineered immunoglobulins and MHC molecules. Useful gene products alsoinclude complement regulatory proteins such as complement regulatoryproteins, membrane cofactor protein (MCP), decay accelerating factor(DAF), CR1, CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation and/or lipid modulation, including the lowdensity lipoprotein (LDL) receptor, high density lipoprotein (HDL)receptor, the very low density lipoprotein (VLDL) receptor, andscavenger receptors. The invention also encompasses gene products suchas members of the steroid hormone receptor superfamily includingglucocorticoid receptors and estrogen receptors, Vitamin D receptors andother nuclear receptors. In addition, useful gene products includetranscription factors such as jun, fos, max, mad, serum response factor(SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins,TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1,CCAAT-box binding proteins, interferon regulation factor (IRF-1), Wilmstumor protein, ETS-binding protein, STAT, GATA-box binding proteins,e.g., GATA-3, and the forkhead family of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,cystathione beta-synthase, branched chain ketoacid decarboxylase,albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methylmalonyl CoA mutase, glutaryl CoA dehydrogenase, insulin,beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin gene product [e.g., a mini- or micro-dystrophin]. Still otheruseful gene products include enzymes such as may be useful in enzymereplacement therapy, which is useful in a variety of conditionsresulting from deficient activity of enzyme. For example, enzymes thatcontain mannose-6-phosphate may be utilized in therapies for lysosomalstorage diseases (e.g., a suitable gene includes that encodingβ-glucuronidase (GUSB)).

Still other useful gene products include those used for treatment ofhemophilia, including hemophilia B (including Factor IX) and hemophiliaA (including Factor VIII and its variants, such as the light chain andheavy chain of the heterodimer and the B-deleted domain; U.S. Pat. No.6,200,560 and U.S. Pat. No. 6,221,349). The Factor VIII gene codes for2351 amino acids and the protein has six domains, designated from theamino to the terminal carboxy terminus as A1-A2-B-A3-C1-C2 [Wood et al,Nature, 312:330 (1984); Vehar et al., Nature 312:337 (1984); and Tooleet al, Nature, 342:337 (1984)]. Human Factor VIII is processed withinthe cell to yield a heterodimer primarily comprising a heavy chaincontaining the A1, A2 and B domains and a light chain containing the A3,C1 and C2 domains. Both the single chain polypeptide and the heterodimercirculate in the plasma as inactive precursors, until activated bythrombin cleavage between the A2 and B domains, which releases the Bdomain and results in a heavy chain consisting of the A1 and A2 domains.The B domain is deleted in the activated procoagulant form of theprotein. Additionally, in the native protein, two polypeptide chains(“a” and “b”), flanking the B domain, are bound to a divalent calciumcation.

In some embodiments, the minigene comprises first 57 base pairs of theFactor VIII heavy chain which encodes the 10 amino acid signal sequence,as well as the human growth hormone (hGH) polyadenylation sequence. Inalternative embodiments, the minigene further comprises the A1 and A2domains, as well as 5 amino acids from the N-terminus of the B domain,and/or 85 amino acids of the C-terminus of the B domain, as well as theA3, C1 and C2 domains. In yet other embodiments, the nucleic acidsencoding Factor VIII heavy chain and light chain are provided in asingle minigene separated by 42 nucleic acids coding for 14 amino acidsof the B domain [U.S. Pat. No. 6,200,560].

As used herein, a therapeutically effective amount is an amount of AAVvector that produces sufficient amounts of Factor VIII to decrease thetime it takes for a subject's blood to clot. Generally, severehemophiliacs having less than 1% of normal levels of Factor VIII have awhole blood clotting time of greater than 60 minutes as compared toapproximately 10 minutes for non-hemophiliacs.

The present invention is not limited to any specific Factor VIIIsequence. Many natural and recombinant forms of Factor VIII have beenisolated and generated. Examples of naturally occurring and recombinantforms of Factor VII can be found in the patent and scientific literatureincluding, U.S. Pat. Nos. 5,563,045; 5,451,521, 5,422,260; 5,004,803;4,757,006; 5,661,008; 5,789,203; 5,681,746; 5,595,886; 5,045,455;5,668,108; 5,633,150; 5,693,499; 5,587,310; 5,171,844; 5,149,637;5,112,950; 4,886,876; International Patent Publication Nos. WO 94/11503,WO 87/07144, WO 92/16557, WO 91/09122, WO 97/03195, WO 96/21035, and WO91/07490; European Patent Application Nos. EP 0 672 138, EP 0 270 618,EP 0 182 448, EP 0 162 067, EP 0 786 474, EP 0 533 862, EP 0 506 757, EP0 874 057,EP 0 795 021, EP 0 670 332, EP 0 500 734, EP 0 232 112, and EP0 160 457; Sanberg et al., XXth Int. Congress of the World Fed. OfHemophilia (1992), and Lind et al., Eur. J. Biochem., 232:19 (1995).

Nucleic acids sequences coding for the above-described Factor VIII canbe obtained using recombinant methods or by deriving the sequence from avector known to include the same. Furthermore, the desired sequence canbe isolated directly from cells and tissues containing the same, usingstandard techniques, such as phenol extraction and PCR of cDNA orgenomic DNA [See, e.g., Sambrook et al]. Nucleotide sequences can alsobe produced synthetically, rather than cloned. The complete sequence canbe assembled from overlapping oligonucleotides prepared by standardmethods and assembled into a complete coding sequence [See, e.g., Edge,Nature 292:757 (1981); Nambari et al, Science, 223:1299 (1984); and Jayet al, J. Biol. Chem. 259:6311 (1984).

Furthermore, the invention is not limited to human Factor VIII. Indeed,it is intended that the present invention encompass Factor VIII fromanimals other than humans, including but not limited to companionanimals (e.g., canine, felines, and equines), livestock (e.g., bovines,caprines and ovines), laboratory animals, marine mammals, large cats,etc.

The AAV vectors may contain a nucleic acid coding for fragments ofFactor VIII which is itself not biologically active, yet whenadministered into the subject improves or restores the blood clottingtime. For example, as discussed above, the Factor VIII protein comprisestwo polypeptide chains: a heavy chain and a light chain separated by aB-domain which is cleaved during processing. As demonstrated by thepresent invention, co-transducing recipient cells with the Factor VIIIheavy and light chains leads to the expression of biologically activeFactor VIII. Because most hemophiliacs contain a mutation or deletion inonly one of the chains (e.g., heavy or light chain), it may be possibleto administer only the chain defective in the patient to supply theother chain.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene is particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce “self”-directed antibodies. Tcell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors (TCRs) that bind to endogenousantigens and initiate the inflammatory cascade associated withautoimmune diseases.

C. Immunogenic Transgenes

Suitably, the AAV vectors of the invention avoid the generation ofimmune responses to the AAV sequences contained within the vector.However, these vectors may nonetheless be formulated in a manner thatpermits the expression of a transgene carried by the vectors to inducean immune response to a selected antigen. For example, in order topromote an immune response, the transgene may be expressed from aconstitutive promoter, the vector can be adjuvanted as described herein,and/or the vector can be put into degenerating tissue.

Examples of suitable immunogenic transgenes include those selected froma variety of viral families. Examples of desirable viral familiesagainst which an immune response would be desirable include, thepicornavirus family, which includes the genera rhinoviruses, which areresponsible for about 50% of cases of the common cold; the generaenteroviruses, which include polioviruses, coxsackieviruses,echoviruses, and human enteroviruses such as hepatitis A virus; and thegenera apthoviruses, which are responsible for foot and mouth diseases,primarily in non-human animals. Within the picornavirus family ofviruses, target antigens include the VP1, VP2, VP3, VP4, and VPG. Otherviral families include the astroviruses and the calcivirus family. Thecalcivirus family encompasses the Norwalk group of viruses, which are animportant causative agent of epidemic gastroenteritis. Still anotherviral family desirable for use in targeting antigens for inducing immuneresponses in humans and non-human animals is the togavirus family, whichincludes the genera alphavirus, which include Sindbis viruses, RossRivervirus, and Venezuelan, Eastern & Western Equine encephalitis, andrubivirus, including Rubella virus. The Flaviviridae family includesdengue, yellow fever, Japanese encephalitis, St. Louis encephalitis andtick borne encephalitis viruses. Other target antigens may be generatedfrom the Hepatitis C or the coronavirus family, which includes a numberof non-human viruses such as infectious bronchitis virus (poultry),porcine transmissible gastroenteric virus (pig), porcinehemagglutination encephalomyelitis virus (pig), feline infectiousperitonitis virus (cat), feline enteric coronavirus (cat), caninecoronavirus (dog), and human respiratory coronaviruses, which may causethe common cold and/or non-A, B or C hepatitis, and which include theputative cause of sudden acute respiratory syndrome (SARS). Within thecoronavirus family, target antigens include the E1 (also called M ormatrix protein), E2 (also called S or Spike protein), E3 (also called HEor hemagglutin-elterose) glycoprotein (not present in allcoronaviruses), or N (nucleocapsid). Still other antigens may betargeted against the Arterivirus family and the rhabdovirus family. Therhabdovirus family includes the genera vesiculovirus (e.g., VesicularStomatitis Virus), and the general lyssavirus (e.g., rabies). Within therhabdovirus family, suitable antigens may be derived from the G proteinor the N protein. The family filoviridae, which includes hemorrhagicfever viruses such as Marburg and Ebola virus may be a suitable sourceof antigens. The paramyxovirus family includes parainfluenza Virus Type1, parainfluenza Virus Type 3, bovine parainfluenza Virus Type 3,rubulavirus (mumps virus, parainfluenza Virus Type 2, parainfluenzavirus Type 4, Newcastle disease virus (chickens), rinderpest,morbillivirus, which includes measles and canine distemper, andpneumovirus, which includes respiratory syncytial virus. The influenzavirus is classified within the family orthomyxovirus and is a suitablesource of antigen (e.g., the HA protein, the N1 protein). The bunyavirusfamily includes the genera bunyavirus (California encephalitis, LaCrosse), phlebovirus (Rift Valley Fever), hantavirus (puremala is ahemahagin fever virus), nairovirus (Nairobi sheep disease) and variousunassigned bungaviruses. The arenavirus family provides a source ofantigens against LCM and Lassa fever virus. Another source of antigensis the bornavirus family. The reovirus family includes the generareovirus, rotavirus (which causes acute gastroenteritis in children),orbiviruses, and cultivirus (Colorado Tick fever, Lebombo (humans),equine encephalosis, blue tongue). The retrovirus family includes thesub-family oncorivirinal which encompasses such human and veterinarydiseases as feline leukemia virus, HTLVI and HTLVII, lentivirinal (whichincludes HIV, simian immunodeficiency virus, feline immunodeficiencyvirus, equine infectious anemia virus, and spumavirinal).

With respect to HIV and SIV, many suitable antigens have been describedand can readily be selected. Examples of suitable HIV and SIV antigensinclude, without limitation the gag, pol, Vif, Vpx, VPR, Env, Tat andRev proteins, as well as various fragments thereof. For example,suitable fragments of the envelope (env) protein include, e.g., gp41,gp140, and gp120. In addition, a variety of modifications to these andother HIV and SIV antigens have been described. Suitable antigens forthis purpose are known to those of skill in the art. For example, onemay select a sequence encoding the gag, pol, Vif, and Vpr, Env, Tat andRev, amongst other proteins. See, e.g., the modified gag protein whichis described in U.S. Pat. No. 5,972,596. See, also, the HIV and SIVproteins described in D. H. Barouch et al, J. Virol., 75(5):2462-2467(March 2001), and R. R. Amara, et al, Science, 292:69-74 (6 Apr. 2001).These proteins or subunits thereof may be delivered alone, or incombination via separate vectors or from a single vector.

The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). The adenovirus family includesviruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/orenteritis. The parvovirus family includes feline parvovirus (felineenteritis), feline panleucopeniavirus, canine parvovirus, and porcineparvovirus. The herpesvirus family includes the sub-familyalphaherpesvirinae, which encompasses the genera simplexvirus (HSVI,HSVII), varicellovirus (pseudorabies, varicella zoster) and thesub-family betaherpesvirinae, which includes the genera cytomegalovirus(HCMV, muromegalovirus) and the sub-family gammaherpesvirinae, whichincludes the genera lymphocryptovirus, EBV (Burkitts lymphoma), humanherpesviruses 6A, 6B and 7, Kaposi's sarcoma-associated herpesvirus andcercopithecine herpesvirus (B virus), infectious rhinotracheitis,Marek's disease virus, and rhadinovirus. The poxvirus family includesthe sub-family chordopoxyirinae, which encompasses the generaorthopoxvirus (Variola major (Smallpox) and Vaccinia (Cowpox)),parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus,and the sub-family entomopoxyirinae. The hepadnavirus family includesthe Hepatitis B virus. One unclassified virus which may be suitablesource of antigens is the Hepatitis delta virus, Hepatitis E virus, andprions. Another virus which is a source of antigens is Nipan Virus.Still other viral sources may include avian infectious bursal diseasevirus and porcine respiratory and reproductive syndrome virus. Thealphavirus family includes equine arteritis virus and variousEncephalitis viruses.

The present invention may also encompass immunogens which are useful toimmunize a human or non-human animal against other pathogens includingbacteria, fungi, parasitic microorganisms or multicellular parasiteswhich infect human and non-human vertebrates, or from a cancer cell ortumor cell. Examples of bacterial pathogens include pathogenicgram-positive cocci include pneumococci; staphylococci (and the toxinsproduced thereby, e.g., enterotoxin B); and streptococci. Pathogenicgram-negative cocci include meningococcus; gonococcus. Pathogenicenteric gram-negative bacilli include Enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (which causes chancroid); brucellaspecies (brucellosis); Francisella tularensis (which causes tularemia);Yersinia pestis (plague) and other yersinia (pasteurella);streptobacillus moniliformis and spirillum; Gram-positive bacilliinclude listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracis(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism (Clostridun botulinum and its toxin); Clostridium perfringensand its epsilon toxin; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude glanders (Burkholderia mallei); actinomycosis; nocardiosis;cryptococcosis, blastomycosis, histoplasmosis and coccidioidomycosis;candidiasis, aspergillosis, and mucormycosis; sporotrichosis;paracoccidiodomycosis, petriellidiosis, torulopsosis, mycetoma andchromomycosis; and dermatophytosis. Rickettsial infections includeTyphus fever, Rocky Mountain spotted fever, Q fever (Coxiella burnetti),and Rickettsialpox. Examples of mycoplasma and chlamydial infectionsinclude: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis;and perinatal chlamydial infections. Pathogenic eukaryotes encompasspathogenic protozoans and helminths and infections produced therebyinclude: amebiasis; malaria; leishmaniasis; trypanosomiasis;toxoplasmosis; Pneumocystis carinii; Trichans; Toxoplasma gondii;babesiosis; giardiasis; trichinosis; filariasis; schistosomiasis;nematodes; trematodes or flukes; and cestode (tapeworm) infections.

Many of these organisms and/or the toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the vectors of the invention to deliver immunogensagainst the variable region of the T cells elicit an immune responseincluding CTLs to eliminate those T cells. In rheumatoid arthritis (RA),several specific variable regions of TCRs which are involved in thedisease have been characterized. These TCRs include V-3, V-14, V-17 andV-17. Thus, delivery of a nucleic acid sequence that encodes at leastone of these polypeptides will elicit an immune response that willtarget T cells involved in RA. In multiple sclerosis (MS), severalspecific variable regions of TCRs which are involved in the disease havebeen characterized. These TCRs include V-7 and V-10. Thus, delivery of anucleic acid sequence that encodes at least one of these polypeptideswill elicit an immune response that will target T cells involved in MS.In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V-6,V-8, V-14 and V-16, V-3C, V-7, V-14, V-15, V-16, V-28 and V-12. Thus,delivery of a nucleic acid molecule that encodes at least one of thesepolypeptides will elicit an immune response that will target T cellsinvolved in scleroderma.

Thus, a rAAV-derived recombinant viral vector of the invention providesan efficient gene transfer vehicle which can deliver a selectedtransgene to a selected host cell in vivo or ex vivo even where theorganism has neutralizing antibodies to one or more AAV sources. In oneembodiment, the rAAV and the cells are mixed ex vivo; the infected cellsare cultured using conventional methodologies; and the transduced cellsare re-infused into the patient.

These compositions are particularly well suited to gene delivery fortherapeutic purposes and for immunization, including inducing protectiveimmunity. AAV of the invention and compositions containing same can alsobe used in immunization regimens such as those described in co-ownedU.S. Patent Application No. 60/565,936, filed Apr. 28, 2004 for“Sequential Adenovirus and AAV-Mediated Delivery of ImmunogenicMolecules”.

Further, the compositions of the invention may also be used forproduction of a desired gene product in vitro. For in vitro production,a desired product (e.g., a protein) may be obtained from a desiredculture following transfection of host cells with a rAAV containing themolecule encoding the desired product and culturing the cell cultureunder conditions which permit expression. The expressed product may thenbe purified and isolated, as desired. Suitable techniques fortransfection, cell culturing, purification, and isolation are known tothose of skill in the art.

The following examples illustrate several aspects and embodiments of theinvention.

Example 1

According to the method of the invention, AAV sequences have beenidentified as having singletons, when placed in an alignment with alibrary of sequences containing representatives of each of clades A, B,C, D, E, and F (represented by AAV9). The following table illustratesthe capsid sequences and the singleton to be altered to a conservedsequence. For certain mutations, the singleton is followed by an * andthen the amino acid reside which replaces it. For other mutations, thesingleton is followed by its amino acid position and the residue whichreplaced it.

The amino acid numbering is based upon the published sequences for eachof these AAV capsids. See, e.g., G. Gao, et al., J. Virol.,78(12):6381-6388 (June 2004) and International Patent Publication No. WO2004/042397 [all sequences therein deposited with GenBank], andInternational Patent Publication No. WO 2005/033321, filed Sep. 30,2004, which are incorporated by reference.

For example, with reference to the following table, the nomenclatureshould be read as follow. Cy5R1 refers to the amino acid sequence of SEQID NO. 24, which has been modified to contain an aspartic acid (D) inamino acid residue position 13; cy5 has a glycine in its native aminoacid sequence at residue number 13. Cy5R2 refers to the amino acidsequence of SEQ ID NO:24, which has been modified to contain an asparticacid in amino acid position 13 (glycine in the native sequence) and anasparagine in amino acid residue position 403 (aspartic acid in thenative sequence). Cy5R3 has the amino acid sequence of SEQ ID NO:24,which has been modified to have the same modifications as the Cy5R2 and,additionally, a lysine at position 158 (natively an asparagine) and aglutamine at position 161 (natively a proline). Given this information,one of skill in the art should be readily able to determine the othersingleton modifications recited in the following table.

SEQ ID NO: (Parent Name AAV) Sites Mutated Clade cy5 24 Cy5R1 G13D DCy5R2 G13D D403N D Cy5R3 G13D D403N R51K D Cy5R4 G13D D403N R51K N158K +D P161Q rh.13 26 D RH.13R E538K D Rh37 40 D Rh37R2 E634K T207M D Rh.2 39E rh.2R V651I E Rh.8 41 rh.8R D531E Rh.48 44 Rh.48.1 K217E B Rh.48.2S304N B Rh.48.1.2 K217E S304N B Hu.44 45 A Hu.44R1 E137K A Hu.44R2 E137KP446L A Hu.44R3 E137K P446L G609D A Rh32/33 2 Hu.29 42 B Hu.29R G396E BCh.5 46 Ch.5R1 T611I rh.67 47 D rh.58 48 S653N E Rh.64 43 E Rh64R1 R697WE Rh64R2 R697W V686E E AAV6 29 A AAV6.2 F129L A AAV6.1 K531E A AAV6.12F129L K531E A rh.54 49 V404M D hu.48 50 A hu.48R1 G277S A hu.48R2 G277SE322K A hu.48R3 G277S E322K S552N A

Example 2

In a preliminary study, five clones were selected to test the singletonmethod of the invention. The table below provides the phenotypedescription of the 5 clones. The number of predicted singletons is givenwith the clade and serotype classification.

Packaging phenotype is considered insufficient when their titer is lowerthan 1×10¹¹GC, low when lower than 1×10¹²GC, good when lower 1×10¹³,excellent when higher.

Gene transfer phenotypes were established by CB.A1AT gene expression andindicated as follows; “+++” better than lead candidate for targettissue, “++”, “+” and “−” respectively better than 50%, between 10-50%or lower than 10% of A1AT serum levels of lead candidates (muscle: AAV1,Liver:AAV8, Lung:AAV9). “n/a” indicated that vector could not beproduced at sufficient levels for in vivo gene transfer studies.

Cloning of the singleton corrections went as follows. From the originalpackaging plasmid, site directed mutagenesis was performed. Subsequentto that, vector backbone integrity was assayed by a PstI digest andcorrection of the singleton was confirmed by sequencing. EGFP-expressingvector was then produced in triplicates on 12-well format side by sidewith the parental singleton-containing vector, AAV2 and AAV2/8 positivecontrol and a production without presence of packaging plasmid as anegative control. Equal volume of harvested lysate after a 3× freeze wasincubated on 293 cells. eGFP expressing was monitored by flow cytometry72 h post transduction.

Site-directed mutagenesis of the singleton residues in clones rh.37,rh.2, ch.5, rh.13, and rh.8 was performed. These particular sequenceswere selected to represent a variety of phenotypes that were previouslydocumented.

Packaging Gene Transfer Phenotype Clade Clone Phenotype Lung LiverMuscle # Singleton (serotype) rh.37 insufficient n/a n/a n/a 2 D (AAV7)rh.2 Low ++ + +++ 1 E (AAV8) ch.5 Good − − − 1 Ch.5 rh.13Excellent + + + 1 D (AAV7) rh.8 Good − + ++ 1 Rh.8

An increase in vector expression was noticed for 4 out of 5 clones. Theincrease was most dramatic for rh.37 and rh.2, vectors that werepreviously shown to have a low packaging yield. For these vectorsproductive particles were produced at levels sufficient for detection.Vectors rh.8 and rh.13 showed an increase in transduction.

In order to distinguish the effects of the singleton mutation ontransduction versus packaging and assembly, small-scale vectorpreparations were made and titered for Dnase resistant particles byquantitative PCR. For rh.37, a two-log increase in vector production wasobserved. rh.8 showed a moderate 5 fold increase in titer whereas rh.13performed equally. All titers of singleton corrected clones were withinacceptable range in comparison to AAV2 and AAV2/8 production and whenextrapolated to large-scale preparations. rh.2 was not assayed fortitration.

Subsequently the effect of the singleton change was monitored in vitroin a transduction setting with equal particle number per cell. Atitration on 293 cells was performed for rh.8 and rh.13. Moderateincreases in transduction efficiency were described at all MOIs.

From this initial subset of 5 clones, 3 productively transduced cells.Two clones were unable to yield any eGFP expression in this setting.This is most likely due to a defect in packaging of the vector thatcould not be predicted by the singleton approach.

The method of the invention was utilized to correct four predictedsingleton locations in AAV clone hu. 46, P156S R362C S393F A676.However, these modifications did not result in an AAV which could berescued, indicating another type of fatal error in the hu. 46 sequence.

Example 3 In Vitro Analysis of Viral Vectors with Altered Capsids

Using the methods of the invention, the capsid proteins of rh.64 andhu.29 were altered and then used to construct viral vectors with thealtered capsids using pseudotyping as described in Example 2 and in G.Gao et al., Proc Natl Acad Sci USA 99, 11854-9 (Sep. 3, 2002).

Briefly, vectors expressing enhanced green fluorescent protein (EGFP)were used to examine in vitro transduction efficiency of the vectors inhuman endothelial kidney cells (293 cells). These 293 cells wereincubated in the presence of 10⁴ GC/cell pseudotyped AAVCMVeGFPparticles after a short pre-incubation with wtAd5. The number of eGFPpositive cells per 10,000 total cells was measured by FACS analysis witha limit of detection of 5 cells/10K.

Modification of the Rh.64 capsid according to the invention affordedmodified rh.64 particles which were over a 100-fold more efficient aftera R697W change. A subsequent V686E mutation yielded a 2-fold increase inpackaging capability.

Modification of the Hu.29 capsid according to the invention affordedmodified rh.64 virions that were rescued from a deficient packagingcapability by changing G396E. A greater than 1000-fold increase inproduction was observed.

Many of the over 20 modified AAV virions showed improvement inexpression include, AAV6.1, hu.48R1, hu.48R2, hu.44R2, hu.44R3, rh.48.2,rh.48.2, rh.48.2.1.

Example 4 Singleton Effect in In Vivo Gene Transfer Applications

The effects of the singleton mutants were studied in an in vivo setting.Gene transfers studies on C57B/6 mice have been initiated on a number ofvectors modified according to the method of the invention. Muscledirected and liver directed studies were initiated and benchmarkedversus the current lead candidates for the particular application.

Human α-antitrypsin (A1AT) was selected as a sensitive and quantitativereporter gene in the vectors and expressed under the control ofCMV-enhanced chicken β-actin promoter. Employment of the CB promoterenables high levels of tissue non-specific and constitutive A1AT genetransfer to be achieved and also permits use of the same vectorpreparation for gene transfer studies in any tissue of interest.

Muscle was chosen as a first target tissue. 40 different novel vectors(based on 24 different clones each with their respective singletonmutant(s)) were injected intramuscularly in a hind limb of C57B/6 mice.All experiments were performed with 1×10¹¹GC/animal with a CB.A1ATtransgene cassette. Vectors were each time aliquoted at equal volume (50μl) per mouse and group per Clade. Every individual study comprised oneor two clades with control groups including the representative serotype,AAV2/8 and AAV2/1 that served as benchmarks for muscle targeted genetransfer. Transgene expression is detected at day 7, 14, 28 and 63 postinjection and evaluated by a specific hA1AT ELISA.

For several isolates and singleton corrected versions, data on theirperformance after intraportal liver-directed infusion was generated.Preliminary results show that the majority of the corrected clonesperform equal or better than the original isolated.

For one particular clone namely cy.5, the singleton correction seem tobeneficial effect on muscle transduction. The clone cy.5R4 carrying 4singleton corrections improved gene transfer efficiency on an alreadydecent muscle tropism exhibited by the original isolate. The performanceof cy.5R4 is equal or slightly better than the benchmark controls AAV2/1and AAV2/7.

An isolate that was previously yielded too low titers for furtherevaluation, rh.64, performed exceptionally well in muscle aftercorrection of one singleton, Rh.64R1 performed better than rh64.2 andgave hA1AT levels higher than those achieved by its closest relativeserotype AAV2/8 but also than AAV2/7.

In other studies, mice were injected with vector in groups based on theclades. 1×10¹¹GC/mouse was dosed with vector expressing CB.hA1AT. Serumlevels of hA1AT were measured by specific hA1AT ELISA.

The effects of singleton on in vivo gene transfer seem to be dependenton isolate and target tissue. Several interesting observations weremade.

For certain singleton clones, the effects are qualitatively similar inmuscle and liver (e.g. rh.2, rh.13 or cy.5). Isolates hu.48 and rh.48show an increased expression in muscle with increased number ofsingletons reverted.

Other clones like rh.64 and AAV6 show a particular expression profile.Isolate hu.48R2 for example packages about 10fold less efficient whencompared to hu.48R3 but the latter transduces muscle about 5-fold lessefficient. AAV6 contains two singletons. Both have moderate effects onpackaging and combined they bring AAV6 packaging up to benchmark level.In vitro, little difference is noticeable between the parental clone andthe different clones. In vivo, in muscle, AAV6.1 and AAV6.1.2 showdecreased gene transfer whereas AAV6.2 show a moderate increase.

Example 5 Evaluation of Singleton-Corrected AAV in Lung and Liver

AAV vectors optimized for packaging and gene transfer efficiency by thereversion of singleton residues were further evaluated in lung andliver. The data is presented for both vectors that were identified asnon-singleton containing or for those for which the singleton residuewas converted to the conserved amino-acid.

A. Evaluation of CB.A1AT AAV Gene Transfer to Lung after IntratrachealInjection Mediated by pi2, rh32.33, AAV2/9, AAV2/5, rh.2R, ch5R.

Several AAV capsids are compared in their ability to target lung. hA1ATlevels were measured in serum. The AAVs evaluated are either singletonfree (pi2, rh32.33, AAV2/9, AAV2/5, rh.2R, ch5R) or contain onesingleton residue (rh.2, rh.8). AAV2/5 and AAV2/9 are represented asbenchmarks.

The gene transfer studies were performed in C57B/6 mice (male, 5 pergroup) using the vectors carrying either the CB.A1AT expression cassette(i.e., AAV2 5′ ITR, chicken β-actin promoter (CB), human α1-antitrypsin(A1AT), AAV2 3′ ITR) or the CB.nLacZ expression cassette (i.e., AAV2,5′ITR, nuclear-localized β-galactosidase (nLacZ), AAV2 3′ITR) in thecapsids described above. Briefly, 50 μL of these singleton-corrected orsingleton-free vectors were co-instilled (1×10¹¹ genome copies (GC))intratracheally with vectors carrying the A1AT and the vectors carryingthe nLacZ (1×10¹¹ GC).

At days 12 and 20, 20 bleeds were taken and serum levels of A1AT weremeasured (ng AAT/mL serum). The data showed a dramatic increase of humanα1-antitrypsin expression in lung for rh.2 to rh.2R after intratracheal(IT) injection of 1×10¹¹ GC. In addition a variety AAV vectors that arefree of singleton residues were evaluated. All vectors showed acceptablelevels of expression in lung.

B. Evaluation of AAV6 Singleton Vectors in Comparison to the AAV2/5 andAAV2/9

AAV6 singleton corrected clones were evaluated. Modified AAV6 (AAV6.2)was prepared using the singleton correction method of the invention, andthe pseudotyping techniques described herein. The AAV6.2 particlescarrying A1AT and LacZ expression cassettes, prepared as described inExample 5, were coinjected intranasally (1×10¹¹ GC) and intratracheally.AAT expression was evaluated by ELISA in serum and in bronchial alveolarliquid (BAL). Expression levels were normalized for total protein. LacZexpression was measured by ELISA for β-galactosidase from lunghomogenate. Necropsy was performed at day 21.

These vectors were compared to AAV2/6, a current clinical candidate forlung gene transfer, AAV 2/5 and AAV2/9 in a study involving C57 Bl/6mice (male, n=8/group).

AAV6.2 presented statistically significant improvement over AAV6 inserum A1AT excretion. AAV6.2 also showed higher levels of A1AT levels ascompared to the other vectors, including AAV2/9 and AAV2/5. Mildimprovement in BAL was noted as was for LacZ expression in lunghomogenate. However, due to large animal to animal variations, noconclusions could be drawn from LacZ quantitation.

When evaluating the localization of AAV gene expression, superiorstaining for nuclear localized LacZ in the AAV2/6.2 group was observed,as compared to AAV 2/6. There was marked improvement over AAV2/6 andAAV2/5 in lung airway epithelium, the primary target for diseases likecystic fibrosis.

C. Intraportal (iv) Injection of AAV CB.A1AT (1×10¹¹ GC) in C57Bl/6 Micewith Clade B and Clade C AAV Members.

All vectors used are absent of singleton residues either from isolation(AAV2/8, AAV2, hu.13, hu.51, hu.11, hu.53) or by mutation (hu.29R) Allvectors are compared to AAV2/8 (clade E) as a benchmark.

D. Intravenous Injection of AAV Members of Clade E. rh.64R1, rh.64R2,rh.2R are Singleton Optimized. All Other Vector are Singleton Free.

The expression from AAV Clade B and C members was found similar toequivalent for all members including hu.29R, a singleton optimizedclone. This particular clone was reconstituted in packaging capabilityfrom a hu.29 and now presents similar gene transfer functionality toother members of the virus family.

For Clade E vectors evaluated, all vectors that are either singletonfree naturally or corrected for singleton residues perform in thesimilar range as the current best performer for liver directed genetransfer, AAV2/8. Particularly AAV rh64R1 and rh.64R2 are of interest.rh.64, found to be defective in packaging, now performs equally well inliver directed gene transfer after conversion of one (rh.64R1) or two(rh.64R2) singletons. For rh.2 the singleton correction corresponds to adramatic more than 10 fold increase in gene delivery.

All publications, including patents, cited in this specification areincorporated herein by reference. While the invention has been describedwith reference to particularly preferred embodiments, it will beappreciated that modifications can be made without departing from thespirit of the invention.

1. A method of increasing the packaging yield, transduction efficiency,and/or gene transfer efficiency of a parental AAV, said methodcomprising the steps of: (a) comparing a parental AAV capsid sequencewith a library of functional AAV capsid sequences in an alignment; (b)identifying at least one singleton in the parental AAV capsid, saidsingleton being a variable amino acid in a position in the parental AAVcapsid in which the amino acid in the corresponding position of thealigned functional AAV capsid sequences is completely conserved; (c)altering the singleton to the amino acid located in the correspondingposition in the functional AAV capsid sequences.
 2. The method accordingto claim 1, wherein the library of aligned functional AAV capsidsequences comprises at least 4 AAV sequences which are least 85%identical over the full-length of the aligned vp1 sequences and containsAAV sequences from at least two different AAV clades.
 3. The methodaccording to claim 1, wherein the library comprises at least two AAVsequences from each clade.
 4. The method according to claim 1, whereinthe library of aligned AAV capsid sequences comprises AAV sequences fromat least three different clades.
 5. The method according to claim 1,wherein the library of aligned AAV capsid sequences comprises at leastthree to 100 AAV sequences.
 6. The method according to claim 1, whereinthe library of aligned AAV capsid sequences comprises at least six to 50AAV sequences.
 7. The method according to claim 6, wherein the librarycomprises at least 12 AAV sequences.
 8. The method according to claim 1,wherein the parental AAV contains multiple singletons.
 9. The methodaccording to claim 8, wherein the parental AAV contains 2 to 4singletons.
 10. The method according to claim 8, wherein step (c) isrepeated at different singletons.
 11. The method according to claim 1,wherein the target AAV is altered by site directed mutagenesis of thecodon for the variable amino acid.
 12. The method according to claim 11,wherein the site directed mutagenesis is performed on the target AAVcarried on a plasmid backbone.
 13. The method according to claim 1,further comprising the step of packaging the altered AAV into an AAVparticle.
 14. The method according to claim 13, wherein the AAV particlehas increased packaging efficiency as compared to the target AAV. 15.The method according to claim 13, wherein in the AAV particle hasincreased transduction efficiency as compared to the target AAV.
 16. Themethod according to claim 13, wherein the AAV particle has increasedgene transfer efficiency as compared to the target AAV.
 17. A viralvector comprising an AAV capsid modified according to the method ofclaim
 1. 18. A vector comprising an AAV capsid selected from the groupconsisting of: rh20, which has an amino acid sequence of SEQ ID NO:1 ora sequence having at least 97% identity thereto; rh32/33, which has anamino acid sequence of SEQ ID NO:2 or a sequence having at least 97%identity thereto; rh39, which has an amino acid sequence of SEQ ID NO:3or a sequence having at least 97% identity thereto; rh46, which has anamino acid sequence of SEQ ID NO:4 or a sequence having at least 97%identity thereto; rh73, which has an amino acid sequence of SEQ ID NO:5or a sequence having at least 97% identity thereto; rh74 which has anamino acid sequence of SEQ ID NO:6 or a sequence having at least 97%identity thereto.
 19. The viral vector according to claim 18, whereinsaid viral vector carries a transgene encoding a gene product under thecontrol of regulatory sequences which direct expression of the productin a host cell.
 20. A modified viral vector having an adeno-associatedcapsid protein selected from the group consisting of: AAV6.1, which hasan amino acid sequence of SEQ ID NO: 29 modified by having a glutamicacid at amino acid residue position 531 which is natively a lysine;AAV6.2, which has an amino acid which has an amino acid sequence of SEQID NO: 29 modified by having a leucine at amino acid residue position129 which is natively a phenylalanine; AAV6.1.2, which has an amino acidsequence of SEQ ID NO: 29 modified by having a lysine at amino acidresidue 129 position which is natively a phenylalanine and a glutamicacid at amino acid residue 531 position which is natively a leucine; 21.A viral vector having a modified adeno-associated capsid selected fromthe group consisting of: rh.8R, which has an amino acid sequence of SEQID NO: 41, which is modified to have a glutamic acid in amino acidresidue position 531, which is natively an aspartic acid; rh1.48.1,which has an amino acid sequence of SEQ ID NO: 44, which is modified tohave a glutamic acid at amino acid residue position 217, which isnatively a lysine; rh.48.2, which has an amino acid sequence of SEQ IDNO: 44, which is modified to have an asparagine at amino acid residueposition 304, which is natively a serine; rh.48.1.2, which has an aminoacid sequence of SEQ ID NO: 44, which is modified to have an glutamicacid at amino acid residue position 217, which is natively a lysine andan asparagine at amino acid residue position 304, which is natively aserine; hu.44R1, which has an amino acid sequence of SEQ ID NO: 45,which is modified to have a lysine at amino acid residue position 137,which is natively an glutamic acid; hu.44R2, which has an amino acidsequence of SEQ ID NO: 45, which is modified to have a lysine at aminoacid residue position 137, which is natively an glutamic acid and aleucine at amino acid position 446, which is natively a proline;hu.44R3, which has an amino acid sequence of SEQ ID NO: 45, which ismodified to have a lysine at amino acid residue position 137, which isnatively an glutamic acid and a leucine at amino acid position 446,which is natively a proline, and an aspartic acid at amino acid position609, which is natively a glycine; hu.29R, which has an amino acidsequence of SEQ ID NO: 42, which is modified to have a glutamic acid atamino acid position 396, which is natively a glycine; hu.48R1, which hasan amino acid sequence of SEQ ID NO: 38, which has been modified tocontain a serine at amino acid residue position 277, which is natively aglycine; hu.48R2, which has an amino acid sequence of SEQ ID NO: 38,which has been modified to contain a serine at amino acid residueposition 277, which is natively a glycine and to contain a lysine atamino acid residue position 322, which is natively an glutamic acid; andhu.48R3, which has an amino acid sequence of SEQ ID NO: 38, which hasbeen modified to contain a serine at amino acid residue position 277,which is natively a glycine and to contain a lysine at amino acidresidue position 322, which is natively a glutamic acid, and to containan asparagine at amino acid residue position 552, which is natively aserine. 22-23. (canceled)
 24. A nucleic acid molecule comprising anucleic acid sequence encoding an AAV sequence selected from the groupconsisting of: rh20, which has an amino acid sequence of SEQ ID NO:1;rh32/33, which has an amino acid sequence of SEQ ID NO:2; rh39, whichhas an amino acid sequence of SEQ ID NO:3; rh46, which has an amino acidsequence of SEQ ID NO:4; rh73, which has an amino acid sequence of SEQID NO:5; rh74 which has an amino acid sequence of SEQ ID NO:6; rh54,which has an amino acid sequence of SEQ ID NO:49, modified by having amethionine at amino acid residue position 404, which is natively avaline; AAV6.1, which has an amino acid sequence of SEQ ID NO: 29modified by having a glutamic acid at amino acid residue position 531which is natively a lysine; AAV6.2, which has an amino acid which has anamino acid sequence of SEQ ID NO: 29 modified by having a leucine atamino acid residue position 129 which is natively a phenylalanine;AAV6.1.2, which has an amino acid sequence of SEQ ID NO: 29 modified byhaving a lysine at amino acid residue 129 position which is natively aphenylalanine and a glutamic acid at amino acid residue 531 positionwhich is natively a leucine; rh.8R, which has an amino acid sequence ofSEQ ID NO: 41, which is modified to have a glutamic acid in amino acidresidue position 531, which is natively an aspartic acid; rh.48.1, whichhas an amino acid sequence of SEQ ID NO: 44, which is modified to have aglutamic acid at amino acid residue position 217, which is natively alysine; rh.48.2, which has an amino acid sequence of SEQ ID NO: 44,which is modified to have an asparagine at amino acid residue position304, which is natively a serine; rh.48.1.2, which has an amino acidsequence of SEQ ID NO: 44, which is modified to have an glutamic acid atamino acid residue position 217, which is natively a lysine and anasparagine at amino acid residue position 304, which is natively aserine; hu.44R1, which has an amino acid sequence of SEQ ID NO: 45,which is modified to have a lysine at amino acid residue position 137,which is natively an glutamic acid; hu.44R2, which has an amino acidsequence of SEQ ID NO: 45, which is modified to have a lysine at aminoacid residue position 137, which is natively an glutamic acid and aleucine at amino acid position 446, which is natively a proline;hu.44R3, which has an amino acid sequence of SEQ ID NO: 45, which ismodified to have a lysine at amino acid residue position 137, which isnatively an glutamic acid and a leucine at amino acid position 446,which is natively a proline, and an aspartic acid at amino acid position609, which is natively a glycine; hu.29R, which has an amino acidsequence of SEQ ID NO: 42, which is modified to have a glutamic acid atamino acid position 396, which is natively a glycine; hu.48R1, which hasan amino acid sequence of SEQ ID NO: 4, which has been modified tocontain a serine at amino acid residue position 277, which is natively aglycine; hu.48R2, which has an amino acid sequence of SEQ ID NO: 4,which has been modified to contain a serine at amino acid residueposition 277, which is natively a glycine and to contain a lysine atamino acid residue position 322, which is natively an glutamic acid; andhu.48R3, which has an amino acid sequence of SEQ ID NO: 4, which hasbeen modified to contain a serine at amino acid residue position 277,which is natively a glycine and to contain a lysine at amino acidresidue position 322, which is natively a glutamic acid, and to containan asparagine at amino acid residue position 552, which is natively aserine.
 25. A method of delivering a gene product to a subject, saidmethod comprising delivering the viral vector according to claim 17 tothe subject.